Project Summary Progressive osseous heteroplasia (POH) is a rare genetic disorder of extraskeletal bone formation, or heterotopic ossification (HO). POH first presents in childhood, with HO formation initiating in the skin and subcutaneous white fat, then progressing to deeper tissues, resulting in severely impaired movement and growth retardation. POH is associated with inactivating mutations of the GNAS locus. The major product of this locus, Gs?/Gnas, encodes the alpha stimulatory subunit of the G protein complex, which transmits signals from G-protein coupled receptors (GPCRs) to activate cyclic-AMP (cAMP). Previous data from our lab have shown that decreased Gnas expression in murine adipose derived stromal cells (ASCs) leads to increased osteogenic potential and decreased adipogenic potential in vitro, as well as reduced subcutaneous adipose tissue that is replaced by HO in vivo. These data indicate that the HO development in POH is through a process of misdirected cell fate, however, the specific tissue and signaling mechanisms that initiate the aberrant osteogenic differentiation and maintain HO progression remain poorly understood. Cell fate decisions are influenced in part by the surrounding physical microenvironment, including stiffness of the extracellular matrix (ECM). Cells sense and interpret the physical cues exerted by ECM components through a process known as mechanotransduction. The ECM of adipose tissue is particularly important in the development and maintenance of this tissue. Dynamic ECM changes during early adipogenesis are necessary to support the differentiating adipocyte. GPCR and Gnas/cAMP signaling have been shown to regulate the key mechanotransduction signaling factors RhoA and transcription co-activators YAP/TAZ, suggesting that the inactivating GNAS mutations in POH could modulate mechano-signaling pathways. I hypothesize that GNAS inactivation alters mechanotransduction signaling to promote osteogenesis of adipose derived stromal cells and impairs maintenance of the adipose tissue microenvironment. In this proposal, I will investigate how Gnas inactivation alters the adipose tissue microenvironment and extracellular biomechanical cues that ASCs receive, and modulates intracellular signaling by biomechanical pathways to promote osteogenesis over adipogenesis. Aim 1 will investigate the effects of Gnas inactivation on the stiffness and ECM composition of adipose tissue microenvironment through in vitro assays and targeted mouse models. Aim 2 will determine the influence of the mutant adipose tissue microenvironment on ASC differentiation using in vivo ASC implant studies. Aim 3 will examine biomechanical signaling pathway activity to determine the effects of decreased Gnas expression on the ability of ASCs to properly sense and respond to soft vs. stiff microenvironments. The investigation of rare disorders of HO will advance our understanding of the regulation of cell fate decisions and provide insight into the aberrant mechanisms that cause HO formation.